Abstract
Iron deficiency anaemia (IDA) makes an individual prone to bacterial infections. The antimicrobial defence mechanism of neutrophils is orchestrated by Nicotinamide Adenine Dinucleotide Phosphate Hydrogen (NADPH) oxidative burst which is iron-dependent. The few previous studies documenting a decrease in neutrophil oxidative burst in iron-deficient children have been based mainly on the Nitro blue tetrazolium test (NBT). Very few studies have been conducted using the more robust flow cytometry-based dihydro rhodamine (DHR) assay in this regard worldwide and none in India. Aim: To estimate the effect of iron deficiency on neutrophil oxidative burst activity in children under 5 years of age by flow cytometry-based dihydro rhodamine (DHR) assay and compare it with the control group. Methods: Thirty-six children between 6 months to 5 years of age diagnosed with moderate (Hb 7-10 gm/dl) to severe (Hb <7 gm/dl) iron deficiency anaemia were selected as cases with equal number of sex/age matched controls. The peripheral blood was analyzed for hematological and biochemical parameters such as complete iron profile, serum vitamin B12, and folate levels. The oxidative burst activity of neutrophils in peripheral blood was assessed using a flow-cytometry-based Dihydrorhodamine (DHR) assay. Results: The percentage of neutrophils showing stimulation, Mean Fluorescence Index in stimulated neutrophils, and Neutrophil oxidative index (NOI) were significantly reduced in iron deficiency anaemia patients as compared to controls. In cases, haemoglobin showed significant positive correlation with NOI and percentage of neutrophils showing stimulation. Conclusion: To conclude, a significant decrease in neutrophil oxidative burst parameters depicts an insufficient innate immune response to pathogens and makes Iron deficiency anaemia patients more susceptible to infections, further aggravated by the severity of anaemia.
Keywords: Preschool children, iron deficiency anaemia, neutrophil oxidative burst, flowcytometry, Dihydrorhodamine assay
Introduction
Anaemia is a major public health issue responsible for the loss of approximately 50 million years of healthy life due to disability by anaemia in the year 2019. Globally, 39.8%% of children aged 6-59 months (<5 years age), suffered from anaemia with iron deficiency being the most common and widespread cause of anaemia in children [1,2]. As per National Family Health Survey (NFHS-5), on Indian children from 6-59 months age, overall, 67.1% of children had some degree of anaemia (<11 g/dl) which was higher than that quoted in NFHS-4 [3].
Iron deficiency anaemia is a problem of serious public health significance that affects mental and physical development, homeostasis, and work performance. Iron is a necessary element for the normal development of the immune system having a role in multiple pathways of both Innate and Adaptive immune response. It is documented that the incidence of infections increases in the presence of Iron deficiency anaemia, however the exact underlying mechanism is unknown [4]. Iron deficiency is associated with the impairment of cell-mediated immunity caused by reduced CD4+ lymphocyte levels and low CD4:CD8 ratio. However, the effect of iron deficiency on humoral and innate immunity is less well described than its role in adaptive immunity [5-7].
Neutrophils being one of the important cells of innate immune response act via Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase 2 (NOX2) mechanism and produce reactive oxygen species to protect against microbes. NOX2 is a heterodimeric enzyme complex in the plasma membrane of neutrophils, which has haem moiety containing ferrous Iron in its cytoplasmic subunit [8].
Several studies found that the phagocytic activity of neutrophils is dependent on serum Iron levels and this activity gets reduced in IDA [9-13]. The reason for a reduction in neutrophil oxidative burst activity in IDA can be due to a decrease in the activity of NADPH oxidase which is a heme-dependent enzyme. Depleted Iron stores and reduced plasma Iron in IDA may affect Iron availability for enzyme activity and thus lead to decreased oxidative burst activity. To support this fact, various studies were conducted to demonstrate the association between iron and oxidative burst activity of neutrophils by comparing iron-deficient individuals with healthy controls [9-13].
A recent ex-vivo study demonstrated that plasma iron modulates the phagocytic capacity as the neutrophils produced during hypoferremia exhibited reduced capacity to phagocytose fluorescent E. coli and staphylococcus aureus [9]. Other studies demonstrated significantly lower NADPH oxidase activities in Iron-deficient patients in comparison to healthy controls [4,5,10-13]. Few researchers also found increased oxidative bursts of neutrophils after administration of Iron supplements [4,5].
Various methods can be used to measure NADPH oxidative burst phenomena, such as 3’, 3’-diaminobenzidine (DAB) oxidation, p-nitro blue tetrazolium (NBT) reduction, chemiluminescence of superoxide ions released by neutrophils, Cytochrome c reduction colorimetric assay and the newer flow cytometry-based DHR (Dihydrorhodamine) assay [14-16].
The Chemiluminescence and colorimetric tests are complicated and laborious while NBT is highly subjective and thus prone to inter-observer variability. On the contrary, flow cytometry-based DHR assay (Dihydro rhodamine assay) is more objective, rapid, highly sensitive and specific to assess the oxidative burst activity of neutrophils as it measures the activity of thousands of neutrophils in a short period [17,18]. Few previous studies documenting a decrease in neutrophil oxidative burst in iron-deficient children have been based mainly on the NBT test.
Very few studies have been conducted using the much more robust DHR assay in this regard all over the world and none in preschool children in India [19,20]. Also, no study has been conducted to measure the neutrophil burst activity using DHR assay in preschool children in whom IDA is very rampant. Thus, the present study aims to assess neutrophil oxidative burst activity by DHR assay in children with iron deficiency anaemia less than 5 years of age and compare it with healthy age-matched controls.
Material and methods
It was a prospective, cross-sectional, and comparative study conducted in the Department of Paediatrics and Pathology of a 1500-bed tertiary care hospital in Delhi. Ethical approval for the study was obtained from the Institutional Ethics Committee for human research.
Study subjects
All Children between 6 months to 5 years in age presenting to paediatric out patient department and confirmed to have Iron deficiency anaemia were included for the study. Iron deficiency anaemia was diagnosed on the basis of Hb less than 10 gm/dL, microcytic hypochromic blood picture and a serum Ferritin <12 µg/l and/or Transferrin saturation (TSAT) <16%. Subjects with mild anaemia (Hb 10-10.99 gm/dl), coexisting B12 or Folate deficiency, history of blood transfusion, history of iron intake in the past 3 months, severe malnutrition as per WHO criteria, known cases of HIV/chronic granulomatous disease/primary immunodeficiency disease, children on steroids >4 weeks, immunosuppressant (oral methotrexate and chemotherapy) and children with rheumatological illness/chronic renal failure/chronic liver disease were excluded from the study.
Thus, a total of 36 children with Iron deficiency anaemia were included as cases and were further classified as moderately (Hb 7-9.9 gm/dl) to severely (Hb <7 gm/dl) anemic. An equal number of healthy age and sex matched children presenting to immunization clinic during that period were recruited as controls. A written informed assent was taken from the parents/guardians of the study subjects participating as cases and controls.
Clinical and demographic details
A baseline assessment including clinical history (immunization history and birth history) and examination was done at the time of enrolment. Demographic details including age, sex, address, contact number, date of enrolment and anthropometry (weight, height and body mass index) were recorded in the case record form.
Laboratory assessment
Laboratory assessment was done using peripheral venous blood obtained under all aseptic and universal precautions. 7 ml of peripheral venous blood was drawn under aseptic conditions and aliquoted in one EDTA and one plain vial. 2 ml blood in EDTA vial was used for complete hemogram by automated haematology analyzer (Mindray BC 6800), peripheral smear examination, ESR (fully automated ALIFAX Roller 20 LC ESR analyser) and flow cytometry based DHR assay performed by Beckman coulter cytomics FC500 flow cytometry analyzer. The reagents DHR and PMA were dissolved in DMSO, 5 µl and 10 µl aliquots were prepared and stored at -20°C. The rest 5 mL blood in iron free plain tube was centrifuged to separate the serum and was stored at -20°C, later used for Iron profile (by spectrophotometry), serum vitamin B12, folate and serum ferritin. The serum ferritin, vitamin B12 and folate were estimated using respective commercially available ELISA kits procured from Cal biotech Pvt Ltd.
Iron profile
The parameters of iron profile were measured according to ICSH 1978 guidelines [10]. Serum Iron (SI) levels less than 60 μg/dL, TSAT levels <16% along with a raised TIBC were taken as Iron deficient. The normal values of SI, TSAT and TIBC considered normal in our laboratory are 60-170 μg/dL, 16-50% and 250-400 μg/dL respectively.
Dihydrorhodamine (DHR) assay
Sample preparation
For each case, 2 tubes were prepared, stimulated and unstimulated, and appropriately labelled. Simultaneously, 2 tubes were prepared for control for each case (Figure 1).
Figure 1.
DHR Assay procedure.
Acquisition and analysis
Flowcytometric analysis was carried out on 5 colour 3 laser cytomics FC 500 flow cytometer from Beckman coulter. After excluding the debris, neutrophils were gated on the basis of forward scatter and side scatter characteristics. Care was taken to include only neutrophils by drawing appropriate gates around them. DHR flow histograms on this gated population were analyzed in both cases and controls for the parameters such as Percentage neutrophils that underwent stimulation, Mean fluorescence Intensity (MFI) of stimulated neutrophils, Mean fluorescence Intensity (MFI) of unstimulated neutrophils and Neutrophil oxidative index (NOI = MFI stimulated tube/MFI unstimulated tube).
Statistical analysis
The data was entered into a computer-based spreadsheet and analyzed using SPSS20.0. The neutrophil oxidative burst was estimated by Mean ± Standard Deviation. Student (unpaired) T-test was used for the comparison of hematological parameters, percent stimulated neutrophils, and mean NOI between cases and controls. The correlation of percentage of neutrophil showing stimulation and hemoglobin was done using the Pearson correlation coefficient. The p-value less than 0.05 was considered significant.
Results
The age of the patients ranged from 6 months to 5 years with a mean ± SD of 3.9±1.0 in the case group and 2.4±1.2 in the control group. Out of the 36 cases, 26 cases were moderately anemic and 10 cases were severely anemic.
Hematological parameters
Hemoglobin ranged from 3.3 to 9.9 g/dl with a mean ± SD of 7.7±1.6 g/dl in cases and 11.2 to 13.4 g/dl with mean ± SD of 12.2±0.5 g/dl in controls. The hemoglobin, hematocrit, RBC count, MCV, MCH, MCHC were significantly higher in controls as compared to cases (P=0.000). Platelets were significantly higher in cases as compared to controls (P=0.000) signifying reactive thrombocytosis. ESR was significantly higher in cases as compared to controls (P=0.000). The Total Leucocyte Count (TLC) of cases ranged from 4300-24600 with a Mean ± SD of 12522±5800. In controls, TLC ranged from 4400-13000 with a Mean ± SD of 7102±1695. The difference was statistically significant (P=0.000) (Table 1).
Table 1.
Hematologic parameters in cases and controls
| Parameters | Cases (n=36) | Controls (n=36) | p-value | ||
|---|---|---|---|---|---|
|
|
|
||||
| Mean ± SD | Range | Mean ± SD | Range | ||
| Hb (g/dl) | 7.7±1.6 | 3.3-9.9 | 12.2±0.57 | 11.2-13.4 | 0.000* |
| Hematocrit (%) | 26±4.4 | 14.6-37 | 36.7±3.31 | 26.6-46 | 0.000* |
| RBC count (×1012/L) | 4.1±0.78 | 3-6.4 | 4.4±0.26 | 4-5.2 | 0.052* |
| MCV (fl) | 56.5±7.7 | 43-76 | 83.1±5.4 | 72-94 | 0.000* |
| MCH (pg) | 17±3.6 | 11-23 | 27±2 | 23-31 | 0.000* |
| MCHC (g/dl) | 28±3.9 | 20-36.4 | 32±1.4 | 27.7-36.6 | 0.000* |
| TLC (×109) | 12522±5800 | 4300-24600 | 7102±1695 | 4400-13000 | 0.000* |
| Platelet count (×109) | 4.7±1.8 | 1.9-8.7 | 2.5±1.6 | 1.3-4.4 | 0.000* |
| ESR (mm in 1st hour) | 22±14.9 | 7-60 | 4±2.8 | 2-15 | 0.000* |
Denotes statistically significant p-value.
Iron profile
The Serum Iron and TIBC were significantly lower in cases as compared to controls and the TIBC was significantly higher in the former. Also, comparing cases with moderate and severe anaemia, it was found that Serum Iron was significantly lower in cases with severe anaemia as compared to those with moderate anaemia, though no significant difference was found between TIBC and TSAT between the two groups (Table 2).
Table 2.
Serum iron parameters in cases (n=36)
| Parameters | Total Cases (n=36) | Moderate Anaemia (n=26) | Severe anaemia (n=10) | p-value |
|---|---|---|---|---|
|
| ||||
| Mean ± SD | Mean ± SD | Mean ± SD | ||
| Serum Iron (µg/dl) | 41±23 | 48±16 | 25±7 | 0.001* |
| TIBC (µg/dl) | 439±22.7 | 437±22.7 | 442±23 | 0.567 |
| Transferrin saturation (%) | 8.4±2.4 | 8.5±2.6 | 8.1±2.06 | 0.672 |
Denotes statistically significant p-value.
Neutrophil oxidative burst
1. The percentage of neutrophils showing stimulation ranged from 0.67-99 with a Mean ± SD of 83±27 in cases however in controls it ranged from 85-99 with a mean ± SD of 96±2. It was significantly reduced in cases as compared to controls (P=0.002).
2. The MFI in unstimulated neutrophils was 1.0±0.62 in cases and 0.83±0.37 in controls and no significant difference was seen (P=0.166).
3. The MFI in stimulated neutrophils ranged from 6.6-351 (Mean ± SD of 157±71) in cases and 203-432 (Mean ± SD of 1.29±1.4) in controls. Thus the neutrophil MFI post-stimulation was significantly reduced in cases as compared to controls (P=0.000). On further comparing the Neutrophil oxidative index (NOI), it was found significantly reduced in cases (Mean ± SD of 181±108) as compared to controls (Mean ± SD of 422±167) (P=0.000) (Table 3; Figure 2).
Table 3.
DHR assay in the cases (n=36) and controls (n=36)
| DHR assay parameters | Cases | Controls | p-value | ||
|---|---|---|---|---|---|
|
|
|
||||
| Mean ± SD | Range | Mean ± SD | Range | ||
| % Stimulated neutrophils | 83±27 | 0.67-99 | 96±2 | 85-99 | 0.002* |
| MFI (Unstimulated) | 1.0±0.62 | 0.10-3.12 | 0.83±0.37 | 0.37-1.89 | 0.166 |
| MFI (Stimulated) | 157±71 | 6.6-351 | 305±58 | 203-432 | 0.000* |
| NOI | 181±108 | 16.1-486 | 422±167 | 197-858 | 0.000* |
Denotes statistically significant p-value.
Figure 2.
Box plots demonstrating comparison of Mean proportion of neutrophils showing stimulation (A), Mean fluorescence intensity of unstimulated neutrophils (B), Mean fluorescence Intensity of stimulated neutrophils (C), and Mean NOI (D) in cases and controls.
Further, these parameters were compared in children with moderate and severe anaemia. The percentage of neutrophils showing stimulation, MFI in stimulated neutrophils, and NOI were significantly reduced in cases with severe anaemia as compared to cases with moderate anaemia (P=0.03, P=0.016 and P=0.005) respectively (Table 4; Figure 3).
Table 4.
DHR assay in cases with moderate and severe anaemia
| DHR assay parameters | Moderate Anaemia | Severe Anaemia | p-value | ||
|---|---|---|---|---|---|
|
|
|
||||
| Mean ± SD | Range | Mean ± SD | Range | ||
| % Stimulated neutrophils | 87±20 | 17-99 | 63±42 | 0.67-98.23 | 0.03* |
| MFI (Unstimulated) | 0.9±0.28 | 0.45-1.42 | 1.2±1.09 | 0.1-3.1 | 0.145 |
| MFI (Stimulated) | 173±60 | 67-351 | 107±89 | 0.5-245 | 0.016* |
| NOI | 212±10 | 57-486 | 103±62 | 16-216 | 0.005* |
Denotes statistically significant p-value.
Figure 3.
DHR assay histograms depicting a comparison of Mean Fluorescence Intensity in healthy controls, cases with moderate anaemia and severe anaemia before and after stimulation.
Then the Hemoglobin and Iron levels were correlated with Neutrophil stimulation parameters. The Hemoglobin level showed a significant positive correlation with NOI and the percentage of neutrophils showing stimulation (r=0.514, P=0.001 and r=0.433, P=0.008) respectively (Table 5).
Table 5.
Correlation of hemoglobin with NOI and % stimulated neutrophils in cases
| Neutrophil Oxidative Index (NOI) | % Neutrophils showing stimulation | |||
|---|---|---|---|---|
|
|
|
|||
| r value | p-value | r value | p-value | |
| Hemoglobin | 0.514 | 0.001* | 0.433 | 0.008* |
Denotes statistically significant p-value.
The r value depicts Pearson correlation coefficient.
Discussion
The neutrophil oxidative burst or respiratory burst is a series of reactions in which superoxide ions are generated by the activity of NADPH oxidase (NOX2) and then metabolized to hydrogen peroxide, hypochlorous acid, and other reactive oxygen species to counteract the microbes. It is also a participant in the apoptosis of neutrophils and neutrophil extracellular traps (NETs) [21,22]. NETs play various roles in inflammation, homeostasis and were found to be involved in autoimmunity as well as immuno-thrombotic events in COVID-19 [23-25].
The NOX2 activity is finely tuned and tightly regulated for appropriate immune functions. The lack of functional NADPH oxidase enhances the risk of recurrent infection conversely, excess reactive oxygen species production leads to tissue damage by oxidative stress.
This study was conducted on 36 preschool children with Iron deficiency anaemia and their neutrophil oxidative burst activity was assessed using a flow-cytometry-based DHR assay and was compared with healthy controls. The percentage of neutrophils showing stimulation, MFI before and after stimulation and Neutrophil oxidative index (NOI) was noted for all subjects. The percentage of neutrophils showing stimulation, mean MFI in stimulated neutrophils and NOI was significantly reduced in children with Iron deficiency anaemia as compared to controls. A decrease in these parameters depicts impaired oxidative burst activity in neutrophils in children with IDA. The reason for a reduction in neutrophil oxidative burst activity in IDA can be due to a decrease in the activity of NADPH oxidase which is a heme-dependent enzyme containing iron in its structure. Depleted Iron stores and reduced plasma Iron in IDA may affect Iron availability for enzyme activity and thus lead to decreased oxidative burst activity. Not many studies have been conducted to assess neutrophil function in children using DHR assay. One similar study using a DHR assay demonstrated a significant decrease in oxidative burst activity in neutrophils in children with IDA (P<0.005) in comparison to healthy children [12]. On an extensive search of the literature, we could not find any more studies on children with IDA measuring neutrophil oxidative burst activity using DHR assay. However, there were few studies done on pregnant females and elderly IDA subjects for the assessment of neutrophil oxidative burst activity by DHR assay. The study on elderly IDA subjects showed significantly lower (50%) oxidative burst in IDA in comparison to the control group [26] however study on pregnant females with IDA showed no significant difference [13]. The fact that pregnancy is associated with altered homeostasis and thus the results cannot be compared to children.
Few researchers have used the NBT test to assess neutrophil oxidative burst activity in children with IDA and concluded a significant fall in oxidative burst of neutrophils in children with IDA compared to healthy controls [5,11]. Furthermore, a study by Kurtoglu et al showed significantly lower NADPH oxidase activity in IDA patients than in healthy controls by using a fluorometric procedure [4].
In contrast to our study, there are some studies in which no significant difference was observed in between children with iron deficiency anaemia and healthy controls by NBT [27,28]. Since, these studies were mostly done using the NBT test to assess neutrophil oxidative burst activity, which is very subjective and thus prone to inter-observer variability, the results of our study and these studies cannot be compared. In contrast, the DHR assay is more objective, rapid, and highly sensitive and specific to assess the oxidative burst activity of neutrophils. Therefore, our data on oxidative burst activity in patients and healthy controls in the paediatric population suggests that neutrophil oxidative burst is reduced in IDA.
In the present study neutrophil oxidative burst activity was further lower in children with severe anaemia as compared to those with moderate anaemia (P<0.05). There are very few studies conducted on the association of iron deficiency with neutrophil function and how it varies as the Iron deficiency increases. A study by Chandra et al demonstrated a significant decrease in the oxidative reduction of nitro blue tetrazolium in severe anaemia as compared to moderate anaemia. Also, the bactericidal capacity of neutrophils was significantly lower in children with IDA as compared to healthy controls. The abnormalities in bacterial killing and the NBT were corrected within 4 to 7 days of parenteral iron which implies that iron deficiency is directly proportional to neutrophil function [5]. Thus, similar to our study, this study also demonstrated that neutrophil functions decreased steadily as plasma Iron levels decreased. However, this study was also done using the more subjective NBT assay. Also, the study had fewer subjects as compared to our study. After extensive research, no more studies were found to assess neutrophil oxidative burst in children with moderate and severe IDA.
We found a significant positive correlation of haemoglobin with neutrophil oxidative index and percentages of neutrophil showing stimulation which implies that as haemoglobin levels fall, neutrophil functions further decline. This implies that severely anaemic children are at a higher risk of infections and infection-related morbidity. This is a novel finding of our study as no study could be found correlating these parameters using this highly sensitive and accurate DHR assay. Future large-scale studies are planned to correlate these parameters.
The limitation of our study is that follow-up of cases was not done to find out the effect of iron supplementation on neutrophil oxidative burst. Moreover, this is a hospital-based study with a limited number of patients. Larger community-based studies with more study subjects and follow-up after the correction of Iron deficiency are needed in the future.
Conclusion
The present study found significantly reduced oxidative burst activity of neutrophils in Iron deficient preschool children as compared to healthy age-matched controls. The decrease in these parameters depicts impaired neutrophil function resulting in poor innate response for pathogens thus further emphasizing IDA children are more susceptible to infections. Moreover, the neutrophil activity directly correlated with the severity of anaemia and fall in iron levels. Community-level measures to correct Iron deficiency will have a direct impact on infection-related morbidity in these children.
Acknowledgements
We would like to thank the technical staff of the Pathology Department, University College of Medical Sciences for their constant support and efficient laboratory work.
Disclosure of conflict of interest
None.
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